We are pursuing an HIV vaccine approach based on replication-competent Adenovirus (Ad)-recombinants. The rationale for this strategy is based on the fact that live attenuated vaccines historically have been the most protective, eliciting essentially life-long immunity. Examples include vaccines for small pox, polio, measles, and yellow fever. We conduct pre-clinical vaccine studies in rhesus macaques and challenge with SIV or SHIV (a chimeric SIV virus containing an HIV envelope), viruses that model HIV-infection of humans. We use a prime-boost strategy, first immunizing with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a boosting with HIV/SIV envelope protein. Ad replicates in epithelial cells that line mucosal inductive sites, and therefore elicits strong, persistent cellular immunity at mucosal effector sites as well as in the blood. In studying vaccine-induced cellular immunity, we have recently focused on several specific cell types. Natural killer (NK) cells are a key population which have a rapid response potential, can kill target cells directly, and also mediate antibody-dependent effector functions such as antibody-dependent cellular cytotoxicity (ADCC) which has been shown to contribute to protective efficacy. They also have recently been shown to persist for long periods of time in vivo and to have the capacity to establish immunologic memory. We investigated NK cell and innate lymphoid cell (ILC) dynamics and function in rhesus macaque rectal tissue and blood following mucosal priming with replicating Ad-SIV recombinants, systemic boosting with SIV envelope protein, and subsequent repeated low-dose intravaginal SIV exposures. Mucosal memory-like NK and ILC subsets in rectal and vaginal tissues of chronically infected macaques were also evaluated. Mucosal IL-17+NKp44+ ILCs were previously associated with a decreased risk of SIV acquisition and IFN-g+DN ILCs with an increased risk of acquisition. We confirmed these observations and extended them, showing that not only did NKp44+ and DN cells exhibit similar associations, but the respective effects continued past SIV infection, contributing to regulation of viral loads. The data suggested that mucosal NKp44+ and DN ILC subsets play protective and pathogenic roles contributing to viremia control and disease progression, respectively. We also identified gamma-chain deficient, Syk-deficient memory-like delta-gamma NK cells in rectal, endocervical, ectocervical, and vaginal tissues obtained at necropsy from the rhesus macaques. The overall frequency of the mucosal delta-gamma cell populations was higher in the mucosal tissues of macaques with low VLs compared to macaques with high VLs suggesting there might be a contribution to viremia control. Overall, our results suggested that vaccines that favor expansion of NKp44+ delta-gamma cells, while avoiding DN delta-gamma cells, might be beneficial for control of SIV/HIV infection. Dendritic cells (DCs) are known to orchestrate both innate and adaptive effector immune cell responses. We investigated their role in vaccine-induced immunity in rhesus macaques. Following mucosal immunizations with replicating Ad type 5 host range mutant (Ad5hr)-SIV recombinants, DC subsets and their activation were examined in rectal tissue, blood, and lymph nodes (LN) at 3 timepoints after each immunization. Plasmacytoid DCs, myeloid DCs, and Langerhans cells were significantly increased in the rectal mucosa, but only myeloid DCs were significantly increased in blood post-immunizations. All rectal DC subsets showed increased frequencies of cells expressing activation markers and cytokines post-immunization, blood DCs showed mixed results, and LN DCs showed few changes. Rectal DCs responded strongly to the vector rather than expressed SIV antigens, but rectal DC frequencies positively correlated with induced rectal antigen-specific memory T and B cells. These correlations were confirmed by in vitro co-cultures showing that rectal Ad-SIV DCs induced proliferation and antigen-specific cytokine production by autologous naive T cells. These results highlighted the rapid response of DCs to Ad immunization and their role in mucosal immune activation and identified initial cellular mechanisms of the replicating Ad-SIV vaccine in the rhesus macaque model. Alveolar macrophages (AMs) play a critical role in lung innate immunity. Using the SIV rhesus macaque model, we investigated the effect of SIV infection on the phenotypic and functional properties of AMs. AM expression of proinflammatory cytokines TNF-a, IL-6, IL-1B, and chemokine RANTES drastically increased 2 weeks post-infection (wpi) compared to AMs of uninfected macaques but dropped significantly with progression to chronic infection. Phagocytic activity of AMs 2-and 4-wpi was elevated compared to AMs of uninfected animals but again significantly decreased by 12-wpi. By 20-wpi the ability of AMs from chronically infected animals to perform SIV-specific antibody-dependent phagocytosis (ADP) was also diminished. PD-1 was found to be expressed on AMs and showed a strong trend toward correlation with plasma viral load, indicating that similar to over-expression on T-cells, PD-1 expression on AMs may also be associated with disease progression. Importantly, blockade of PD-1 improved phagocytic function. These findings provide new insight into the dynamics of SIV infection leading to AM dysfunction and alteration of pulmonary innate immunity and suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in HIV-infected individuals. Further studies of cells expressing PD-1, led to identification of a dysfunctional CD8+ T cell population co-expressing CD6 and PD-1 in lymphoid tissues and bronchoalveolar lavage of rhesus macaques chronically infected with SIV. This cell population which was expanded compared to uninfected cells, displayed impaired proliferation cytokine secretion and cytotoxicity. The frequency of the cells positively correlated with viremia. The cells expressed elevated levels of the inhibitory receptor LAG-3 and SHP-2 phosphatase compared to CD6-PD-1+ CD8+ T cells suggesting a mechanism by which CD6 might induce T cell dysfunction. Combined targeting of CD6 and PD-1 in vitro revived CD8+ T cell effector function of splenocytes of infected macaques suggesting a potential therapeutic strategy.